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1. Connexin 46 and connexin 50 gap junction channel properties are shaped by structural and dynamic features of their N‐terminal domains

   https://doi.org/10.1113/JP281339  

   Yue, Benny ; Haddad, Bassam G. ; Khan, Umair ; Chen, Honghong ; Atalla, Mena ; Zhang, Ze ; Zuckerman, Daniel M. ; Reichow, Steve L. ; Bai, Donglin ( May 2021 , The Journal of Physiology)

   Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.

   Recent structural studies by cryo‐electron microscopy have produced high‐resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure–function comparison.

   Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform‐specific differences in Cx46 and Cx50 intercellular channel function.

   We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N‐terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.

   The results of this study establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease‐causing variants.

   Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long‐range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino‐terminal (NT) domain as a major contributor for isoform‐specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46‐50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free‐energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage‐dependent gating (V_jgating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra‐ and inter‐subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract‐linked connexin variants.

    

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2. Modulation of TRPV2 by endogenous and exogenous ligands: A computational study

   https://doi.org/10.1002/pro.4490  

   Feng, Shasha ; Pumroy, Ruth A. ; Protopopova, Anna D. ; Moiseenkova‐Bell, Vera Y. ; Im, Wonpil ( December 2022 , Protein Science)

   Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C‐terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor‐like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.

    

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3. Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

   https://doi.org/10.1085/jgp.202112875  

   Li, Jing ; Shen, Rong ; Rohaim, Ahmed ; Mendoza Uriarte, Ramon ; Fajer, Mikolai ; Perozo, Eduardo ; Roux, Benoît ( August 2021 , Journal of General Physiology)

   C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

    

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4. A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

   https://doi.org/10.1085/jgp.202012607  

   Gaete, Pablo S. ; Lillo, Mauricio A. ; López, William ; Liu, Yu ; Jiang, Wenjuan ; Luo, Yun ; Harris, Andrew L. ; Contreras, Jorge E. ( October 2020 , Journal of General Physiology)

   Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease–causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

    

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5. Distinct lipid bilayer compositions have general and protein-specific effects on K+ channel function

   https://doi.org/10.1085/jgp.202012731  

   Winterstein, Laura-Marie ; Kukovetz, Kerri ; Hansen, Ulf-Peter ; Schroeder, Indra ; Van Etten, James L. ; Moroni, Anna ; Thiel, Gerhard ; Rauh, Oliver ( February 2021 , Journal of General Physiology)

   null (Ed.)

   It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K+ channels to systematically examine the impact of bilayer composition on the pore module of single K+ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K+ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity. 

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